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Oral antiretroviral agents provide life-saving treatments for millions of people living with HIV, and can prevent new infections via pre-exposure prophylaxis1–5. However, some people living with HIV who are heavily treatment-experienced have limited or no treatment options, owing to multidrug resistance6. In addition, suboptimal adherence to oral daily regimens can negatively affect the outcome of treatment—which contributes to virologic failure, resistance generation and viral transmission—as well as of pre-exposure prophylaxis, leading to new infections1,2,4,7–9. Long-acting agents from new antiretroviral classes can provide much-needed treatment options for people living with HIV who are heavily treatment-experienced, and additionally can improve adherence10. Here we describe GS-6207, a small molecule that disrupts the functions of HIV capsid protein and is amenable to long-acting therapy owing to its high potency, low in vivo systemic clearance and slow release kinetics from the subcutaneous injection site. Drawing on X-ray crystallographic information, we designed GS-6207 to bind tightly at a conserved interface between capsid protein monomers, where it interferes with capsid-protein-mediated interactions between proteins that are essential for multiple phases of the viral replication cycle. GS-6207 exhibits antiviral activity at picomolar concentrations against all subtypes of HIV-1 that we tested, and shows high synergy and no cross-resistance with approved antiretroviral drugs. In phase-1 clinical studies, monotherapy with a single subcutaneous dose of GS-6207 (450 mg) resulted in a mean log10-transformed reduction of plasma viral load of 2.2 after 9 days, and showed sustained plasma exposure at antivirally active concentrations for more than 6 months. These results provide clinical validation for therapies that target the functions of HIV capsid protein, and demonstrate the potential of GS-6207 as a long-acting agent to treat or prevent infection with HIV. The small molecule GS-6207, which disrupts the function of the HIV capsid protein, shows potential as a long-acting therapeutic agent for the treatment and prevention of HIV infection.

People living with HIV (PLWH) have expressed concern about the life-long burden and stigma associated with taking pills daily and can experience medication fatigue that might lead to suboptimal treatment adherence and the emergence of drug-resistant viral variants, thereby limiting future treatment options1–3. As such, there is strong interest in long-acting antiretroviral (ARV) agents that can be administered less frequently4. Herein, we report GS-CA1, a new archetypal small-molecule HIV capsid inhibitor with exceptional potency against HIV-2 and all major HIV-1 types, including viral variants resistant to the ARVs currently in clinical use. Mechanism-of-action studies indicate that GS-CA1 binds directly to the HIV-1 capsid and interferes with capsid-mediated nuclear import of viral DNA, HIV particle production and ordered capsid assembly. GS-CA1 selects in vitro for unfit GS-CA1-resistant capsid variants that remain fully susceptible to other classes of ARVs. Its high metabolic stability and low solubility enabled sustained drug release in mice following a single subcutaneous dosing. GS-CA1 showed high antiviral efficacy as a long-acting injectable monotherapy in a humanized mouse model of HIV-1 infection, outperforming long-acting rilpivirine. Collectively, these results demonstrate the potential of ultrapotent capsid inhibitors as new long-acting agents for the treatment of HIV-1 infection. A new compound that inhibits HIV capsid assembly and nuclear transport functions offers potential as a long-acting antiretroviral.

KLF10 is a transforming growth factor (TGF)-β/Smad downstream regulated gene. KLF10 binds to the promoter of target genes and mimics the effects of TGF-β as a transcriptional factor. In our laboratory, we noted that Klf10 deficiency in mice is associated with significant inflammation of the lungs. However, the precise mechanism of this association remains unknown. We previously identified NPRA as a target gene potentially regulated by KLF10 through direct binding; NPRA knockout have known that prevented lung inflammation in a mouse model of allergic asthma. Here, we further explored the regulatory association between KLF10 and NPRA on the basis of the aforementioned findings. Our results demonstrated that KLF10 acts as a transcriptional repressor of NPRA and that KLF10 binding reduces NPRA expression in vitro. Compared with wild-type mice, Klf10-deficient mice were more sensitive to lipopolysaccharide or ovalbumin challenge and showed more severe inflammatory histological changes in the lungs. Moreover, Klf10-deficient mice showed pulmonary neutrophil accumulation. These findings collectively reveal the precise site where KLF10 signaling affects pulmonary inflammation by attenuating NPRA expression. They also verify the importance of KLF10 and atrial natriuretic peptide/NPRA in exerting influences on chronic pulmonary disease pathogenesis.

Anesthetic management during functional endoscopic sinus surgery is aimed at minimizing bleeding and establishing a near-perfect surgical field. We investigated whether deliberate intraoperative hypercapnia and hypocapnia may affect blood loss and quality of surgical field through a proposed modulating effect of different carbon dioxide (CO2) tension levels on nasal vasculature.One hundred and eighty patients were randomly assigned to normocapnia (end-tidal CO2 [ETco2] 37 +/- 2 mm Hg), hypercapnia (ETco2 60 +/- 2 mm Hg), and hypocapnia (ETco2 27 +/- 2 mm Hg) groups. Anesthetic management was with propofol and remifentanil infusions, nitrous oxide, and moderate controlled hypotension. Blood loss and operating conditions were assessed by the surgeon who was blinded to group assignment. Differences among the study groups, the effect of the study group and time on ETco2 levels and hemodynamic variables, and the association of blood loss with surgical covariates were analyzed.There were no differences in blood loss and quality of surgical field among the study groups. Patients in the hypocapnia group demonstrated the highest, and in the hypercapnia group, the lowest, requirements for remifentanil, labetalol, and administration of the antihypertensive medications in general. The computed tomography-graded severity of sinonasal disease and duration of surgery were the only independent predictors of intraoperative blood loss.CO2 management during functional endoscopic sinus surgery does not influence operating conditions or blood loss.

OBJECTIVE: The aim of this study was to further evaluate the safety and effectiveness of balloon catheter devices to dilate obstructed sinus ostia/perform sinusotomy. METHODS: Through a prospective, multicenter evaluation, safety was assessed by rate of adverse events, patency was determined by endoscopic examination, and sinus symptoms were determined by the Sino-Nasal Outcome Test (SNOT 20). RESULTS: At the conclusion of the 24-week analysis, endoscopy determined that the sinusotomy was patent in 80.5% (247 of 307) sinuses and nonpatent in 1.6% (5 of 307), and could not determine ostial patency status in 17.9% (55 of 307). Of the ostia visualized on endoscopy, 98% were patent (247 of 252), while 2% (5 of 252) were considered nonpatent. SNOT 20 scores showed consistent symptomatic improvement over baseline. Revision treatment was required in 3 sinuses (3 of 307 sinuses, 0.98%) in 3 patients (3 of 109 patients, 2.75%). CONCLUSION: Balloon catheter technology appears safe and effective in relieving ostial obstruction. Patients were pleased and indicated that they experienced symptomatic improvement.

Immortalization is a critical event in virus-related oncogenesis. No enough information, however, is currently available to elucidate the changes that occur in cellular molecules during immortalization. To identify potential cellular markers or regulators involving in immortalization, a paired-cell model of primary foreskin keratinocytes (FK) and HPV16 immortalized foreskin keratinocytes were established. Using mRNA differential display, RT-PCR and Northern blot methods, we have identified and confirmed that Dyrk1a (dual-specificity tyrosine-phosphorylated and regulated kinase 1A) is present and increased in HPV16 immortalized cells, but is absent in primary keratinocytes. Moreover, transfection of E7 siRNA oligo into immortalized cells leads to a diminishing E7 expression and the eventual disappearance of Dyrk1a. Similar results of Dyrk1a expressional differences could also be seen when tissue specimens were compared using LCM/real-time PCR and immunohistochemistry analysis; malignant cervical lesions contain significantly more DYRK1A than normal tissue. It was also demonstrated that raised DYRK1A could rearrange the cellular localization of FKHR (forkhead in rhabdomyosarcoma), an apoptosis activator, and suppress BAD. Importantly, this phenomenon can be reversed when endogenous Dyrk1a was knocked down in immortalized cells by RNA interference. These results suggest that the raised Dyrk1a in HPV16 immortalized keratinocytes and cervical lesions may serve as a candidate antiapoptotic factor in the FKHR regulated pathway and initiate immortalization and tumorigenesis gradually. © 2007 Wiley-Liss, Inc.

Background Over the past 20 years, many patients have benefited from endoscopic sinus surgery and its ability to relieve sinus obstruction. However, problems still occur with surgery, thereby leaving room for innovation. Recently, catheter-based technology has provided new options for treating cardiac, vascular, and urologic diseases. We speculated that catheter technology also might offer new treatment options for sinusitis patients. The purpose of this investigation was to explore the feasibility and safety of catheter-based technology to relieve sinus ostial obstruction. Methods Anatomic models and human cadaver specimens were used initially to design and iterate catheters to open sinus ostial drainage pathways. Thereafter, the safety of balloon-catheter dilation was evaluated in six human cadaver heads. CT scan obtained before and after catheter ostial dilation was analyzed for evidence of catheter-induced trauma. Dilated ostia also were examined by endoscopy and gross anatomic dissection for unwanted catheter-induced trauma. Results Catheters successfully dilated 31 of 31 ostia, including 9 maxillary, 11 sphenoid, and 11 frontal ostia/recesses. CT scan, endoscopy, and gross anatomic dissection revealed that such dilation did not cause trauma to surrounding structures such as the orbit or skull base. Mucosal trauma imparted by catheter dilation appeared to be less than that normally seen with standard endoscopic instruments. Conclusion This initial study suggests that catheter technology can be used to dilate sinus ostia safely. Mucosal preservation and ease of use make catheters an attractive minimally invasive treatment strategy. Additional testing in patients is indicated to gain additional safety information and to explore the usefulness of catheter-based technology.

Biologically active, therapeutically useful, DNA binding natural products continue to reveal new paradigms for sequence-selective recognition, to enlist beautiful mechanisms of in situ activation for DNA modification, to define new therapeutic targets, to exploit new mechanisms to achieve cellular selectivity, and to provide a rich source of new drugs. These attributes arise in compact structures of complex integrated function.

Objective Informed consent is essential prior to functional endoscopic sinus surgery (FESS). The content of this discussion is often determined by each surgeon. Many doctors discuss all potential complications. This may provoke anxiety or deter from beneficial surgery. Our goal was to examine the process from the patient's perspective. Study design A list of potential complications was reviewed with FESS patients. Patients were asked questions concerning their consent and the potential complications before and after FESS. Results Both before and after surgery, patients felt that discussion of most potential complications was important. Vision changes and cerebrospinal fluid leak (CSF) received the highest scores. No significant differences were found between primary and revision cases. Conclusion Patients wanted most potential FESS complications to be discussed. Even though this triggered anxiety, they felt it was important to have a thorough disclosure. Findings presented may impact how surgeons counsel patients prior to FESS.

Objectives To evaluate the management of patients with large skull base defects (> 2 cm) and intracranial injury caused by powered endoscopic sinus surgery. Study design and setting All patients treated for postendoscopic sinus surgery skull base injury over a 4-year period were reviewed. Results Three patients with skull base defects greater than 2 cm in size and associated intracranial injury from powered ESS were identified. All patients presented with active cerebrospinal fluid leaks. CT scans showed intracranial injury and pathology reports revealed brain tissue removal. Using image-guided endoscopic techniques, all defects were addressed with multilayer repair. Closure was achieved in all patients on the first attempt, with an average follow-up of 27 months. Conclusions The use of powered instrumentation along the skull base can be dangerous and can result in extensive skull base defects with associated loss of dura and gray matter. Large ethmoid roof defects and significant intracranial injury, however, are not absolute contraindications to endoscopic repair.

Infections in patients with chronic sinusitis after surgery can be difficult to treat. Nebulized antimicrobial therapy was studied as a treatment option.Patients with chronic sinusitis, previous sinus surgery, and an acute infection were offered nebulized antibiotics or standard therapy. Cultures were taken and sensitivity testing was performed. Retrospective chart reviews were also performed.Forty-two patients were included. The most common side effects were sore throat and cough. Symptomatic and endoscopic data before and after nebulized therapy showed a longer infection-free period (average, 17 weeks) compared with standard therapy (average, 6 weeks). Improvements in posterior nasal discharge, facial pain/pressure, and emotional consequences were noted.Nebulized therapy was safe and effective in this cohort. Endoscopy and outcome measure changes showed consistent improvements.A novel therapy for acute sinus infections in patients with chronic sinusitis and previous endoscopic sinus surgery is presented.

The role of the serotonergic system in the pathogenesis of behavioral disorders such as depression, alcoholism, obsessive-compulsive disorder, and violence is not completely understood. Measurement of the concentration of neurotransmitters and their metabolites in cerebrospinal fluid (CSF) is considered among the most valid, albeit indirect, methods of assessing central nervous system function in man. However, most studies in humans have measured lumbar CSF concentrations only at single time points, thus not taking into account rhythmic or episodic variations in levels of neurotransmitters, precursors, or metabolites. We have continuously sampled lumbar CSF via subarachnoid catheter in 12 healthy volunteers, aged 20-65 years. One ml (every 10 min) CSF samples were collected at a rate of 0.1ml/min for 24-hour (h), and the levels of tryptophan (TRP) and 5-hydroxy indoleacetic acid (5-HIAA) were measured. Variability across all 12 subjects was significantly greater (P < 0.0001) than the variability seen in repeated analysis of a reference CSF sample for both 5-HIAA (32.0% vs 7.9%) and TRP (25.4% vs 7.0%), confirming the presence of significant biological variability during the 24-hr period examined. This variability could not be explained solely by meal related effects. Cosinor analysis of the 24-hr TRP concentrations from all subjects revealed a significant diurnal pattern in CSF TRP levels, whereas the 5-HIAA data were less consistent. These studies indicate that long-term serial CSF sampling reveals diurnal and biological variability not evident in studies based on single CSF samples.

TGF-β plays a significant role in regulating pancreas islet function and maintaining their mass. KLF10, a TGF-β downstream gene, belongs to a group of Kruppel-like transcription factors that bind to the promoters of target genes and produce effects that mimic TGF-β as a tumor suppressor. Using ChIP-chip screening, SEI-1 was identified as a target gene that may be regulated by KLF10. We conducted a series of assays to verify the presence of unknown regulation events between SEI-1 and KLF10. These showed that KLF10 transcriptionally activates the SEI-1 promoter and, furthermore, induces SEI-1 protein expression in pancreatic carcinoma cells. SEI-1 is one of the key factors involved in cell cycle control through the regulation of other transcription factors such as the p21(Cip1) gene. Interestingly, it has been shown previously that p21(Cip1) is indirectly activated by KLF10. Our results first demonstrated that KLF10 acts as a transcriptional activator on SEI-1, which can then result in increased p21(Cip1) expression. Furthermore, KLF10-deficiency in mice is associated with a decrease in the pancreatic islet mass, which is similar to the effects found in SEI-1 deficient mice. The KLF10-defect was also associated with the nuclear accumulation of the p21(Cip1) in islet cells. Based on our molecular and histological findings, we conclude that KLF10 plays an important role in pancreatic β-cells and this supports a functional link between KLF10 and various cell cycle regulators, most notably in the context of the pancreas.

PURPOSE This study compared the clinical efficacy of oxycodone hydrochloride controlled-release (CR) tablets administered every 12 hours with immediate-release (IR) oxycodone tablets administered four times daily in patients with cancer-related pain. PATIENTS AND METHODS Cancer patients who required therapy for moderate to severe pain were randomized to CR oxycodone every 12 hours (n=81) or IR oxycodone four times daily (n=83) for 5 days in a multicenter, double-blind study. Pain intensity was assessed four times daily (categorical scale of none, slight, moderate, and severe); acceptability of therapy was assessed twice daily (categorical scale of very poor, poor, fair, good, and excellent). RESULTS Pain intensity remained slight during the study, with mean oxycodone doses of 114 mg/d (range, 20 to 400 mg/d) for CR and 127 mg/d (range, 40 to 640 mg/d) for IR. Acceptability of therapy was fair to good with both treatments. While standard conversion ratios provided an acceptable dose for many patients, a protocol amendment that allowed initial titration and use of rescue medication reduced the discontinuation rate for lack of acceptable pain control (from 34% to 4% with CR and from 31% to 19% with IR before and after amendment, respectively) without increasing the discontinuation rate for adverse events (from 8% to 7% with CR and from 13% to 11% with IR). Fewer adverse events were reported with CR (109) than with IR (186) oxycodone (P=.006). CONCLUSION CR oxycodone every 12 hours was as effective as IR oxycodone four times daily in managing moderate to severe cancer-related pain and was associated with fewer reports of adverse events.

Epstein—Barr virus (EBV) induces human B cell transformation and is closely associated with naso-pharyngeal carcinoma. The expression of an EBV latent membrane protein, LMP-1, protects B cells from apoptosis by up-regulating the expression of a cellular oncogene, bcl-2. LMP-1 also transforms rodent fibroblasts and affects the differentiation, morphology and growth of human and rodent epithelial cells. In this report, we describe a novel finding that high level expression of the LMP-1 gene in a human epithelial cell line (RHEK-1) induces apoptosis, characterized by chromosomal DNA fragmentation in the transfected cells. In particular, such an effect was more apparent under serum starvation. We also found that in the transfected RHEK-1 cells, LMP-1 expression neither affected bcl-2 expression nor led the cells to grow in semisolid soft agar medium. These results indicate that LMP-1 may participate in the development of EBV-associated epithelial malignancy via a mechanism different from that seen in B cell or fibroblast transformation.

Cells of a breast adenocarcinoma cell line, CAMA-IEe, were found to contain a moderate concentration of 1,25-dihydroxyvitamin D3[1,25(OH)2D3] receptors. The receptors' dissociation constant calculated by Scatchard analysis was 4.1 x 10(-10) M. These receptors sedimented at around 4.0 S position on a linear 5-20% sucrose density gradient and exhibited an apparent molecular weight of about 46 kDa. Receptor concentration was found to be highest in the early stages of cell growth, decreased continuously when cells were actively growing, and reached the lowest level at confluence. Suppression of cell growth, of DNA synthesis, and of the phosphorylation of retinoblastoma protein by 1,25(OH)2D3 at concentrations about 10(-7) M were observed. Treatment with 10(-7) M 1,25(OH)2D3 for 12 hr or longer caused elevation of cytosolic free calcium in CAMA-IEe cells. These results show that CAMA-IEe cell growth is suppressed by 1,25(OH)2D3 and that the suppression is possibly mediated through calcium mobilization.

GS-9190 (Tegobuvir) is a novel imidazopyridine inhibitor of hepatitis C virus (HCV) RNA replication in vitro and has demonstrated potent antiviral activity in patients chronically infected with genotype 1 (GT1) HCV. GS-9190 exhibits reduced activity against GT2a (JFH1) subgenomic replicons and GT2a (J6/JFH1) infectious virus, suggesting that the compound's mechanism of action involves a genotype-specific viral component. To further investigate the GS-9190 mechanism of action, we utilized the susceptibility differences between GT1b and GT2a by constructing a series of replicon chimeras where combinations of 1b and 2a nonstructural proteins were encoded within the same replicon. The antiviral activities of GS-9190 against the chimeric replicons were reduced to levels comparable to that of the wild-type GT2a replicon in chimeras expressing GT2a NS5B. GT1b replicons in which the β-hairpin region (amino acids 435 to 455) was replaced by the corresponding sequence of GT2a were markedly less susceptible to GS-9190, indicating the importance of the thumb subdomain of the polymerase in this effect. Resistance selection in GT1b replicon cells identified several mutations in NS5B (C316Y, Y448H, Y452H, and C445F) that contributed to the drug resistance phenotype. Reintroduction of these mutations into wild-type replicons conferred resistance to GS-9190, with the number of NS5B mutations correlating with the degree of resistance. Analysis of GS-9190 cross-resistance against previously reported NS5B drug-selected mutations showed that the resistance pattern of GS-9190 is different from other nonnucleoside inhibitors. Collectively, these data demonstrate that GS-9190 represents a novel class of nonnucleoside polymerase inhibitors that interact with NS5B likely through involvement of the β-hairpin in the thumb subdomain.

Background An increasing number of patients seeking care by an otolaryngologist are undergoing computed tomography (CT) examinations via in-office CT scanners. Many otolaryngologists and patients are not fully aware of the radiation dosages and the associated risks. A recent study of common CT examinations demonstrated significant variability in radiation dosages for similar studies. Despite the relatively low doses associated with sinus scans, widely publicized studies and events have generated a renewal of physician, public, and regulatory agency awareness and concern regarding medical radiation exposure. Methods Phantom measurements and/or radiation dosage reports from CT scanners utilized by a large otolaryngology group in California were reviewed and compared. Different types of CT scanners from multiple manufacturers were included. Results There was nearly a 10-fold difference (0.15–1.45 mSv) in radiation between scans obtained from in-office, hospital-based, and outpatient imaging facility CT scanners. Conclusion Significant relative variability can exist in radiation dosages associated with routine sinus CT scans obtained in different locations. There is a need for increased awareness and understanding among otolaryngologists and their patients regarding medical radiation exposure. All otolaryngologists should constantly consider the principle of “as low as reasonably achievable” (ALARA). Shielding, pediatric protocols, and other dosage reduction measures should be utilized whenever possible. © 2011 ARS-AAOA, LLC.

Estrogen stimulates cell growth and inhibits apoptosis through estrogen receptor-mediated mechanisms in many cell types. Remarkably, there is another dimension to estrogen action by which apoptosis is induced in breast cancer cells. While these mechanisms are not yet completely understood, finding the molecules involved has paved the way for the development of a new drug group. Using ChIP-chip, we have demonstrated that Klf10, a Kruppel-like zinc finger transcription factor, which was induced in response to estrogen, directly modulates the transcription of BI-1 (Bax inhibitor-1; also called the testis-enhanced gene transcript, TEGT). Eventually, the estrogen induced Klf10 and then suppresses BI-1 transcription. The estrogen/Klf10/BI-1 interrelationship was further confirmed using BI-1 promoter and EMSA assays. The estrogen-elicited reduction of BI-1 promoter activity was significantly reversed when the Klf10 binding element was mutated to abolish Klf10 binding. A si-Klf10 antisense-oligo nucleotide was also able to restore BI-1 promoter activity to its pre-estrogen-treatment level. BI-1 is known to regulate stress via the endoplasmic reticulum; in this context down-regulation of BI-1 is able to cause Ca(2+) release and trigger an apoptosis pathway in breast cancer. In our study, Klf10 not only suppressed cellular BI-1 expression but also increased the cytosolic Ca(2+) concentration, eventually causing apoptotic cell death. Based on these results, we suggest the pathway by which estrogen induces apoptosis is possibly through an up-regulation of Klf10 that decreases BI-1 and finally increases the concentration of cytoplasmic calcium.

In contrast to HIV infection in humans and SIV in macaques, SIV infection of natural hosts including sooty mangabeys (SM) is non-pathogenic despite robust virus replication. We identified a novel SM CCR5 allele containing a two base pair deletion (Δ2) encoding a truncated molecule that is not expressed on the cell surface and does not support SIV entry in vitro. The allele was present at a 26% frequency in a large SM colony, along with 3% for a CCR5Δ24 deletion allele that also abrogates surface expression. Overall, 8% of animals were homozygous for defective CCR5 alleles and 41% were heterozygous. The mutant allele was also present in wild SM in West Africa. CD8+ and CD4+ T cells displayed a gradient of CCR5 expression across genotype groups, which was highly significant for CD8+ cells. Remarkably, the prevalence of natural SIVsmm infection was not significantly different in animals lacking functional CCR5 compared to heterozygous and homozygous wild-type animals. Furthermore, animals lacking functional CCR5 had robust plasma viral loads, which were only modestly lower than wild-type animals. SIVsmm primary isolates infected both homozygous mutant and wild-type PBMC in a CCR5-independent manner in vitro, and Envs from both CCR5-null and wild-type infected animals used CXCR6, GPR15 and GPR1 in addition to CCR5 in transfected cells. These data clearly indicate that SIVsmm relies on CCR5-independent entry pathways in SM that are homozygous for defective CCR5 alleles and, while the extent of alternative coreceptor use in SM with CCR5 wild type alleles is uncertain, strongly suggest that SIVsmm tropism and host cell targeting in vivo is defined by the distribution and use of alternative entry pathways in addition to CCR5. SIVsmm entry through alternative pathways in vivo raises the possibility of novel CCR5-negative target cells that may be more expendable than CCR5+ cells and enable the virus to replicate efficiently without causing disease in the face of extremely restricted CCR5 expression seen in SM and several other natural host species., Author Summary SIV causes AIDS in macaques, like HIV-1 does in humans, but not in its natural host species such as sooty mangabeys (SM). It is therefore important to understand infection in natural hosts, including the mechanisms and cellular targets of infection. SIV replication in SM is thought to exclusively use CCR5 as its entry coreceptor, which mediates viral entry in conjunction with CD4 and is the main determinant of target cell tropism. However, other molecules also function as SIV coreceptors in vitro. We discovered that inactivating mutations in the CCR5 gene are common among SM and, furthermore, homozygous mutant animals lacking functional CCR5 still become infected and have high viral loads. Ex vivo, SM lymphocytes can be infected independently of CCR5, and several alternative entry coreceptors are used by SIV from both CCR5+ and CCR5-null animals. Thus, SIV infection in SM is mediated by other coreceptors in addition to CCR5, suggesting that these molecules together may determine tropism and cell targeting in vivo. These results provide new insight into an important model of nonpathogenic natural host infection, and identify a novel role for alternative entry pathways suggesting a potentially broader range of target cells in vivo than currently recognized.

The human papillomavirus (HPV) oncoprotein E7 is a major transforming protein. The E7 protein does not possess intrinsic enzymatic activity, but rather functions through direct and indirect interactions with cellular proteins, several of which are well known cellular tumor suppressors. Using the yeast two-hybrid system, we found that transforming growth factor-beta inducible early gene 1 (TIEG1), a member of the Kruppel-like family (KLF) that has been implicated as a putative tumor suppressor, interacts and forms a specific complex with HPV-16 E7. TIEG1 has been shown to mimic the effects of TGF-beta in various carcinoma cells and plays a critical role in the apoptotic cascade. Our results indicate that E7 binds to the C-terminus of TIEG1 and induces its degradation via the ubiquitin pathway. E7 not only increased the ubiquitination of TIEG1 but also influenced the ability of TIEG1 to affect apoptosis. Our results suggest that suppression of TIEG1-mediated signaling by E7 may contribute to HPV-associated carcinogenesis.

The first US Food and Drug Administration-approved dilating balloon catheter system for obstructed paranasal sinus drainage pathways was introduced in September 2005. It was discussed as an alternative treatment option for traditional endoscopic sinus surgery for those suffering from chronic rhinosinusitis. Widespread patient interest has ensued, although controversy regarding application of this device continues. Many otolaryngologists have been trained to use the device, and more than 88,000 patients have had surgery using this device. Like similar dilating catheters used in other specialties to relieve obstruction, it is a minimally invasive tool for mucosal-sparing dilation of sinus ostia or sinus outflow tracts. Although studies have been completed that demonstrate feasibility, safety, and long-term patency of dilated sinuses, the evolving indications for its use remain controversial. Referring physicians and patients can expect a range of opinions on the role of these devices in the treatment of rhinosinusitis for the foreseeable future.

There is a dearth of writings about early detection of potential suicide patients in chronic pain centers. Early detection measures used at the Vanderbilt Pain Control Center include a Symptom Checklist-90, with questions about depressive symptomatology and "Thoughts of Ending Your Life"; medical and psychological interviews; monitoring of changes in emotional disturbance; and, if warranted, administration of the Scale of Suicidal Ideation. Three case studies are presented that indicate that the results of an assessment measure should be tempered with clinical judgment. Suicidal behavior, including suicidal ideation, is a medical emergency; therefore, there is great need for early detection and triage measures. Language: en

This study investigated the extent to which habitual cigarette smoking relates to physical and psychological indices of chronic pain. From a review of patient records, 54% of back pain patients referred for treatment of their pain admitted to smoking cigarettes. Response from a smoking questionnaire showed that 57% of the patients who smoked reported having a need to smoke when they were in pain. Most patients (91%), however, believed that smoking had no effect on their pain intensity. When smoking and nonsmoking back pain patients were compared, the smokers showed significantly higher levels of emotional distress, they tended to remain inactive, and they relied on medication more often than the nonsmoking patients. The results further suggest that pain patients are at risk for increasing smoking behavior when they are experiencing periods of heightened pain intensity.

Assess two-year postoperative clinical outcomes for patients receiving balloon catheter sinusotomy.Patients who had sinus ostia dilated with balloon catheters were prospectively evaluated two years after surgery by Sinonasal Outcome Test (SNOT-20) and computed tomographic (CT) scan.Sixty-five patients (195 ballooned sinuses) were followed for two years after surgery, including 34 "balloon-only" patients and 31 "hybrid" patients. SNOT-20 symptom scores were significantly improved from baseline (0.87 vs 2.17 baseline, P0.001) and stable compared to six months and one year; this was the case for both balloon-only (1.09 vs 2.09, P0.001) and hybrid (0.64 vs 2.26, P0.001) patients. Lund-MacKay CT scores were significantly improved from baseline (2.69 vs 9.66, P0.001) and stable compared to one year, confirmed for both balloon-only (1.75 vs 5.67, P0.015) and hybrid (3.25 vs 12.05, P0.001) subsets of patients. A total of 85% of patients reported improvement of their sinus symptoms, with 15% same and 0% worsened. Revision treatment was required in seven of 195 sinuses (3.6%) in six of 65 patients (9.2%).Patients who receive balloon catheter sinusotomy in endoscopic sinus surgery have significant improvement in symptoms two years after surgery. Radiographic evidence also confirms resolution of disease after two years. This demonstrates durability of clinical results previously reported at 24 weeks and one year after surgery.

HPV-16E7 is a major transforming protein, which has been implicated in the development of cervical cancer. The stability of E7 is thus important to ensure its fully functional status. Using the yeast two-hybrid system, we found that USP11 (ubiquitin-specific protease 11), a member of a protein family that cleaves polyubiquitin chains and/or ubiquitin precursors, interacts and forms a specific complex with HPV-16E7. Our results indicate that the USP11 can greatly increase the steady state level of HPV-16E7 by reducing ubiquitination and attenuating E7 degradation. In contrast, a catalytically inactive mutant of USP11 abolished the deubiquitinating ability and returned E7 to a normal rate of degradation. Moreover, USP11 not only protected E7 from ubiquitination but also influenced E7 function as a modulator of cell growth status. These results suggest that USP11 plays an important role in regulating the levels of E7 protein and subsequently affects the biological function of E7 as well as its contribution to cell transformation by HPV-16E7.

HPV16, a high-risk tumorigenic virus, has been identified as one of the causative agents for the development of cervical cancer. Subsequent to viral infection, the constitutive expression of the viral oncoproteins E6 and E7 plays a number of critical roles in maintaining the transformed phenotype. Here we demonstrate that a cellular kinase, dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), interacts with and phosphorylates HPV16E7 in vitro and in vivo. Using substitution mutations, we identified that DYRK1A specifically phosphorylates HPV16E7 at Thr5 and Thr7, which are located within the N-terminal CRI domain. This interaction greatly increases the steady-state level of HPV-16E7 by interfering with the protein's 26S proteosome-dependent degradation. The half-life of E7 was extended significantly by replacing Thr5 and Thr7 with a phosphorylation mimetic residue, aspartic acid. In addition, DYRK1A-induced phosphorylation protected E7 from degradation and influenced E7's function when modulating pRb degradation. We propose a new mechanism whereby DYRK1A phosphorylates Thr5 and Thr7 within HPV16E7. This phosphorylation then interferes with the degradation of HPV16E7, extending the protein half-life of HPV16E7 and increasing the colony-formation efficacy of HPV16E7. Our findings suggest that DYRK1A increases the transforming potential of HPV16-infected cells because of the greater stability of HPV16E7.

ObjectiveThe primary objective was to assess the long-term effectiveness of balloon catheter sinusotomy.MethodsPatients who had sinus ostia dilated with balloon catheters were prospectively evaluated 1 year after surgery with nasal endoscopy, a CT scan, and the Sino-Nasal Outcome Test (SNOT-20).ResultsSixty-six patients (202 sinuses) were examined. One hundred seventy-two of 202 sinus ostia (85%) were endoscopically patent, 1 percent (2/202) were nonpatent, and ostial patency could not be determined by endoscopy in 28 of 202 (14%). In these “indeterminate” sinuses, the CT scans were normal in 13, implying functional patency in 91.6 percent of sinuses (185/202). Sinus CT scan scores were 1.95 at 1 year versus 8.89 at baseline (P < 0.001), and 1-year SNOT-20 scores (0.91) were significantly improved from baseline (2.14, P < 0.0001).ConclusionBalloon catheter sinusotomy results were durable over the study period, showing long-term effectiveness.

Purpose of review Sinus surgery tools are constantly being developed. This review will focus on new devices (popularly called sinuplasty) for use in ostial dilation. Recent findings Balloons for sinus ostial dilation are approved by the Food and Drug Administration and are used worldwide. There have been presentations, publications and discussions concerning these devices. One large level-2 evidence study has demonstrated these devices to be well tolerated and reliable. Summary Sinuplasty may represent an exciting and minimally invasive set of devices. They may also represent a premature attempt to transfer dilation into otolaryngology. The emerging data suggest the first statement, with excellent outcomes. More long-term data could still prove the second statement correct. Research and development will make these devices a 'hot topic' in sinus surgery for 2007-2008.

This study examined the effect of significant weight gain on physical, demographic, behavioral, and psychosocial factors in a representative sample of chronic pain patients. One hundred fifty-five chronic pain patients who reported gaining more than 15 pounds since the onset of their pain were compared with 341 pain patients who stated that their weight had remained the same since the onset of their pain. All patients were given a medical examination and each patient completed a comprehensive pain questionnaire and an SCL-90. Results showed that a significant relationship exists between weight gain and decreased physical activity, increased emotional distress, and accident liability. This study suggests that the inclusion of weight management training in multidisciplinary pain centers may play an important part in the rehabilitation of chronic pain patients.

Objective The aim of this study was to determine the extent of radiation exposure to the patient and the surgeon in balloon catheter dilation. Methods Dosimeters were used to record radiation exposure to both patients and surgeons. The mean radiation dose per sinus treated and per patient treated was calculated for each dosimeter location. Results The mean patient dose (108 sinuses treated in 34 patients), was 0.32 mSv per sinus and 1.02 mSv per patient over the eye and 1.33 mSv per sinus and 4.22 mSv per patient over the temple. The average total fluoroscopy time was 3.6 minutes per patient. The average number of sinuses treated was 3.2. The mean surgeon dose at the chest (254 sinuses in 89 patients) was 0.025 mSv per sinus and 0.072 mSv per patient. The mean surgeon dose at the hand (182 sinuses in 68 patients) was 0.009 mSv per sinus and 0.023 mSv per patient. Conclusions The use of fluoroscopy in balloon catheter dilation exposes both the patient and surgeon to very low doses of radiation.

Several reports in the literature have indicated that the E6 not only elevates the level of c-Myc level but that the protein also associates with the Myc complex and activates Myc-responsive genes. There would seem to be a mechanism by which this oncogene can modulate cell proliferation and differentiation. Furthermore, an increase in c-Myc levels has also observed during ectopic expression of HPV E7 alone. Using the yeast two-hybrid system, we further found that the c-Myc interacts and forms a specific complex with HPV-16E7. In this study, we have demonstrated that E7 does indeed interact with c-Myc and a sequential deletion analysis of E7 maps the c-Myc interaction site to the carboxyl-terminal region. We determined two HPV-18 E7 binding sites on c-Myc involving the amino acids regions 1-100 and 367-439. The interaction of the high-risk type HPV E7 with c-Myc can augment c-Myc transactivation activity but this does not occur with low-risk type HPV E7. Deletion within the Cys-X-X-Cys repeat motif at the C-terminus of HPV-18 E7 leads to a lost of association with c-Myc and also abolishes the enhancement of c-Myc's transactivation activity. Furthermore, the interaction of HPV-18 E7 with c-Myc functionally promotes c-Myc's DNA-binding ability. Using the hTERT promoter as a model, enhanced c-Myc binding ability to the hTERT promoter as measured by immunoprecipitation assay was observed and occurred in an E7 dose-dependent manner. Taken together, these results provide significant new insights into the association of c-Myc with E7 and the possible involvement of high-risk E7 in oncogenesis.

To evaluate impressions and usage of sinus surgery image-guided surgical (IGS) systems by ENT surgeons in the United States.A survey was mailed to selected practicing ENT physicians, investigating regional information, practice type, IGS usage patterns, perceived benefits and limitations, and usage of the 61795 surgery code.Seventy-three percent of respondents use IGS. Nonusers respond that it provides no benefit or is too expensive. Eighty percent of respondents replied that IGS may allow for increased safety in certain procedures. Most users attempt reimbursement with the 61795 code.IGS usage is increasing but appears to be perceived as expensive and nonbeneficial in certain situations. Most respondents, however, felt that IGS may lead to safer surgery in certain situations, including revision and frontal procedures. Several factors appear to limit routine use including ease of use, technical setup, code reimbursement, and initial purchase costs.IGS use appears to be increasing. The most frequent users appear to agree with the previously issued AAO-HNS guidelines regarding appropriate indications. Expanded use may depend on ease of use, reimbursement, and affordability.D-5.

Based on the RNAi technique, we have developed a new approach that generates transgenic animals capable of mimicking human genetic diseases. The new system is a combination of siRNA with Cre-loxP and tetracycline-on. It has the characteristics of being stable, inheritable, and inducible, with the siRNA able to be transcribed tissue specifically. To support the ability of this new method to generate a model for a disease, we created an ABCA1-deficient mouse line that mimics Tangier disease under controlled conditions. Thus, it should now be possible to rapidly establish human genetic diseases as a whole animal model without the use of embryonic stem cell and gene targeting. This system also provides a tool for pathological and pharmacological studies of aspects peculiar to particular human genetic diseases.

Masses that radiographically appear in the lateral aspect of the frontal sinus can be difficult to access and often are approached through external approaches. Supraorbital ethmoid cells pneumatize the orbital plate of the ethmoid bone to lie posterior and lateral to the frontal sinus. Opacification of a supraorbital cell may radiographically give the appearance of a laterally based frontal sinus lesion. Often, these represent mucoceles, in which their drainage can be achieved through endoscopic techniques and without the need for an external approach.Retrospective review of patients treated for lateral frontal sinus lesions at a tertiary sinus center was performed. Radiology, endoscopic findings, operative reports, and patient symptoms were reviewed.Ten patients were identified with lateral frontal sinus lesions based on radiography of the paranasal sinuses and nasal endoscopy. All patients were determined to be supraorbital mucoceles. These patients underwent surgical drainage using computer-aided endoscopic techniques. Initially, endoscopic drainage of the mucocele was successful in all patients. One patient was lost to follow-up after 3 months and one patient underwent a revision endoscopic surgery with trephination 5 months after the initial drainage. The remaining eight patients remain free of disease by nasal endoscopy and postoperative computed tomography scans (median follow-up of 25 months; range, 8-38 months).Knowledge of the anatomy of the ethmoid complex and presence of various cells within the frontal recess such as supraorbital cells are important in the management of the laterally based frontal sinus lesion. Often, these lesions may represent supraorbital cell mucoceles amenable to endoscopic drainage. Given the nature of mucoceles, long-term follow-up is needed before endoscopic drainage of these lesions can be validated. However, preliminary data suggest that an endoscopic approach provides for adequate drainage and helps avoid external approaches.

Background Revision surgery of the frontal sinus remains one of the most difficult operations for the endoscopic surgeon. Most agree that knowledge and recognition of its complex anatomy and sparing of frontal recess mucosa are keys to a successful operation. The use of surgical navigation systems may allow for more precise dissections and greater rates of frontal recess patency. Methods Retrospective review of all patients undergoing revision endoscopic frontal sinus surgery with surgical navigation was performed with a minimum 24-month follow-up. Results Sixty-seven patients underwent revision endoscopic frontal sinus surgery with surgical navigation. The average follow-up was 32 months. Fifty-eight (86.6%) had a patent frontal recess and significant subjective improvement in symptoms. No patient underwent external frontal sinus obliteration, and there were no major complications. Conclusions Endoscopic techniques with surgical navigation are effective in revision frontal sinus cases. The dissection of remnant agger nasi, obstructing frontal and supraorbital cells are necessary to widen the anterior-posterior as well as the medial-lateral dimensions of the recess. Computer navigational systems appear to serve as a valuable adjunct in preoperative planning and safe intraoperative dissection.

With the aim of identifying potential cellular proteins that mediate the transcriptional regulation of YY1, a HeLa cDNA library was screened using the yeast two-hybrid system. A previously unknown protein interacting with YY1 was identified and named YY1AP. By using the 5'-rapid amplification of cDNA ends technique, the full-length cDNA of YY1AP was cloned and sequenced. The cDNA was 2253 bp in length and encoded an open reading frame of 750 amino acids. The chromosomal gene was made up of 10 exons separated by nine introns and is localized on chromosome 1 (1q21.3). Northern blot analysis revealed that YY1AP is ubiquitously expressed in various human tissues and cancer cell lines. Co-immunoprecipitation and immunostaining of cells further indicated that YY1AP co-localizes with YY1 in the nucleus. Furthermore, YY1AP was shown to be capable of enhancing the transcriptional activation of an YY1 responsive promoter. Subsequent analysis by glutathione S-transferase pull-down assay showed that YY1AP contained two YY1 binding regions. The transactivation region of YY1AP would seem to be localized within the section of amino acids 260-345. It is proposed that YY1AP is a novel co-activator of YY1.

Yin Yang 1 (YY1) is a highly conserved and multifunctional transcription factor. The diverse activities of YY1 are regulated and sometimes modified by interaction with various other proteins. By using a yeast two-hybrid screening system, SAP30 was identified as a protein that associates with YY1 and it is able to enhance YY1-mediated repression in a dose-dependent manner. SAP30 is a 30 kDa nuclear protein and is a component of the human histone deacetylase complex. In this study, the interaction of SAP30 and YY1 was confirmed both by in vitro and in vivo assays. The interaction domains between YY1 and SAP30 were mapped to the C-terminal segment of YY1 (295–414) and the C-terminal 91 amino acid region of SAP30. The observation that YY1, SAP30, and HDAC1 form a complex in vivo provides evidence that YY1 also recruits HDAC1 indirectly via its binding to SAP30. These results describe a novel mechanism for YY1-mediated repression.

Co-expression of B-cell leukemia-lymphoma 2 gene (Bcl-2) and Epstein-Barr virus latent membrane protein 1 (LMP-1) in nasopharyngeal carcinoma tissues were tested using immunohistochemical methods. Results showed that there were 32% (14/44) and 68% (30/44) LMP-1 and Bcl-2-positive cases, respectively. Among the LMP-1-positive tissues, 8 (57%) of 14 specimens were also Bcl-2-positive. The level of LMP-1 and Bcl-2 expression was associated with the clinical stages of nasopharyngeal carcinoma. Furthermore, when LMP-1- and Bcl-2-positive cases were combined, the highest positive score was found in clinical stage II as well as in the early stage (stages I and II) of nasopharyngeal carcinoma. While further studies with more cases are needed, this study suggests that co-expression of Bcl-2 and LMP-1 may be involved in the process of nasopharyngeal carcinoma aggravation.

Difficulty breathing after upper airway surgery requires immediate evaluation and treatment. We present a case of airway compromise after sinus surgery due to edema of the uvula. The patient was admitted for observation overnight and discharged the next day. A discussion of potential airway changes after sinonasal surgery is presented.

In all patients and volunteers, the levels of immunoreactive SP measured in saliva were about 100 times higher than the levels measured in plasma. SP per mg protein was consistently lower in both plasma and saliva of chronic pain patients than in healthy volunteers. These findings suggest that a simple noninvasive objective method of determining SP in saliva may become useful in the evaluation and treatment of chronic pain.

Human brain development is slow and delicate, involving many unique, though interrelated, cellular events. The fetus and child are often more susceptible to chemical toxins that alter the structure and/or function of the brain, although susceptibility varies for individual neurotoxicants. Early exposure to neurotoxins has been implicated in neurological diseases and mental retardation. Pesticide exposures pose a particular concern since many are designed to be neurotoxic to pests and can also affect humans. Acknowledging the potential for vulnerability of the developing brain, EPA recently began to “call in” data on developmental neurotoxicity (DNT) from manufacturers of pesticides already registered and considered to be neurotoxic—around 140 pesticides. Chemicals are to be tested following the DNT testing guideline (OPPTS 870.6300). This paper assesses whether tests performed according to this guideline can effectively identify developmental neurotoxicants. We found the testing guideline deficient in several respects, including: It is not always triggered appropriately within the current tiered system for testing; It does not expose developing animals during all critical periods of vulnerability; It does not assess effects that may become evident later in life; It does not include methodology for consideration of pharmacokinetic variables; Methodology for assessment of neurobehavioral, neuropathological, and morphometry is highly variable; Testing of neurochemical changes is limited and not always required. We propose modifications to the EPA testing guideline that would improve its adequacy for assessing and predicting risks to infants and children. This paper emphasizes that deficiencies in the testing methodology for developmental neurotoxicants represent a significant gap and increase the uncertainty in the establishment of safe levels of exposure to developing individuals.

This study sought to examine the feasibility of prolonged assessment of acetylcholinesterase (AChE) activity in the cerebrospinal fluid (CSF) of volunteers and to test the hypothesis that rivastigmine (ENA-713; Exelon, Novartis Pharma AG, Basel, Switzerland) selectively inhibits AChE in CSF in humans at a dose producing minimal inhibition of the peripheral enzyme. Lumbar CSF samples were collected continuously (0.1 mL x min(-1)) for 49 hours from eight healthy volunteers who took either placebo or a single oral dose of rivastigmine (3 mg). CSF specimens and samples of blood cells and blood plasma were analyzed at intervals for rivastigmine and its metabolite NAP 226-90 ([-] [3-([1-dimethylaminolethyl)-phenol]), erythrocyte AChE activity, CSF AChE activity, and plasma and CSF butyrylcholinesterase (BuChE) activity. Safety evaluations were performed 23 hours after drug dosing and at the end of the study. Evaluable data were obtained from six subjects. The mean time to maximal rivastigmine plasma concentration (tmax) was 0.83 +/- 0.26 hours, the mean maximal plasma concentration (Cmax) was 4.88 +/- 3.82 ng x mL(-1), the mean plasma area under the concentration versus time curve (AUC0-infinity) was 7.43 +/- 4.74 ng x hr x mL(-1), and the mean plasma t1/2 was 0.85 +/- 0.115 hours. The concentration of rivastigmine in CSF was lower than the quantification limit for assay (0.65 ng x mL(-1)), but NAP 226-90 reached a mean Cmax of 3.14 +/- 0.57 ng x mL(-1). Only minimal inhibition of erythrocyte AChE activity (approximately 3%) was observed. Inhibition of AChE in the CSF after rivastigmine administration was significantly greater than after placebo for up to 8.4 hours after the dose and was maximal (40%) at 2.4 hours. Plasma BuChE activity was significantly lower after rivastigmine than after placebo, but this was not clinically relevant. BuChE activity in CSF was significantly lower after rivastigmine than after placebo for up to 3.6 hours after dosing, but this difference was not sustained. This study confirms the feasibility of using continuous measurement of AChE activity in CSF over prolonged periods, that rivastigmine markedly inhibits CSF AChE after a single oral dose of 3 mg, and that the inhibition of central AChE is substantially greater than that of peripheral AChE or BuChE.

A 32-year-old man with chronic intractable right lower extremity pain unresponsive to multiple neurosurgical and pharmacologic treatments, including intrathecal morphine administration, was successfully treated with sciatic nerve block, discontinuance of opioid therapy, and psychologic interventions. Plasma and urine ratios of morphine metabolites morphine-3-glucuronide and morphine-6-glucuronide were analyzed at the beginning of our interventions, and the results indicated that morphine-3-glucuronide levels were significantly higher than morphine-6-glucuronide levels. The possible association between the observed morphine metabolite ratio and the intractable pain in patients resistant to opioids may have potential clinical implications.

Commercial sources for neuropeptide radioimmunoassays have made this sensitive tool available to clinical investigators for monitoring the potential involvement of neuropeptides in pain modulation. We measured substance P-like immunoreactivity in the plasma, saliva, and pericardial fluid of subjects with and without pain (chronic and acute) to determine if substance P levels are altered. Some recent studies have suggested that substance P in various body fluids may be a correlate of chronic pain. To test this correlation it is important to ensure that the assay is measuring what it was designed to measure. Therefore, the influence of three tachykinins on the analysis of substance P concentrations was assessed with a commercially available radioimmunoassay kit. A small (approximately 2 to 6%), apparently nonspecific elevation in measured substance P was found when alpha-neurokinin, beta-neurokinin, or eledoisin was incubated with substance P and its antibody. Our results also indicate an apparent specific affinity of the substance P antibody for alpha-neurokinin (above 1,000 pg/ml) and beta-neurokinin (above 5,000 pg/ml). Substance P levels in the body fluids we tested ranged from 0.47 to 62.88 pg/mg protein (47.4 to 230.8 pg/ml). Levels of the tested tachykinins have not been determined in body fluids. If alpha-neurokinin or beta-neurokinin is found to be present in high concentrations in these fluids, this commercially available substance P kit may overestimate substance P levels. The concentrations of tachykinins necessary to interfere specifically with the assay are 10- to 100-fold higher than substance P in body fluids. If the other tachykinins are present at concentrations similar to the substance P levels, we would not expect them to interfere substantially with the accuracy of our measurements of substance P using this assay.

The in vitro effects of two metabolites of inhalational anaesthetics, fluoride and bromide, on pseudocholinesterase (PCHE) and acetylcholinesterase (ACHE) activities in the blood samples of seven healthy patients were studied. The PCHE and ACHE activities were determined by kinetic spectrophotometric methods. Fluoride at the levels achieved with clinical concentrations of enflurane and sevoflurane (25-75 microM.L-1) inhibited PCHE activity by 28-65 per cent (P less than 0.01) and ACHE activity by less than five per cent (P greater than 0.05). Bromide at the levels achieved with clinical concentrations of inhalational anaesthetics had no significant effect on either PCHE or ACHE activity. We recommend caution when succinylcholine and/or ester type local anaesthetics are used in the immediate postoperative period following enflurane or sevoflurane anaesthesia. We also recommend that blood drawing for PCHE activity be delayed at least until 24 hr following enflurane or sevoflurane anaesthesia.